Enhancement of antibody-cytokine fusion protein mediated immune responses by combined treatment with immunocytokine uptake enhancing agents

ABSTRACT

Disclosed are methods and compositions for treating tumors. Disclosed methods and compositions enhance the uptake of immunocytokines into tumors, and are based on a combination of an immunocytokine with an immunocytokine uptake enhancing agent. Disclosed methods and compositions are particularly useful for reducing tumor size and metastasis in a mammal.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/896,909, filed Jun. 29, 2001, which claims priority to, and thebenefit of 60/215,038 filed Jun. 29, 2000, the disclosures of each ofwhich are incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to antibody-cytokine fusion proteinsuseful for targeted immune therapy. In general, the invention relates tothe use of immunocytokine uptake enhancing agents in combination therapyto enhance an antibody-cytokine fusion protein mediated immune responseagainst a preselected target, for example, cells in a tumor. Inparticular, the invention relates to the administration ofantibody-cytokine fusion proteins in combination with chemotherapeuticssuch as taxanes and/or alkylating agents to treat tumor cells and othercancerous or diseased cells.

BACKGROUND OF THE INVENTION

Effective treatment of diseases such as cancer require robust immuneresponses by one or more effector cell types such as natural killer (NK)cells, macrophage and T lymphocytes. In animals and patients bearingtumors, the immune system has not effectively dealt with the growingtumor due, in large part, to specific mechanisms the tumor haselaborated to suppress the immune response. In many cases, potentiallytumor-destructive monocytic cells, e.g. macrophages, migrate intogrowing tumor beds, but the secretion of factors such as prostaglandins,TGF-β and IL-10 by the tumor cells modulate their cytotoxic activity(see, for example, Sharma et al., 1999, J. IMMUNOL. 163:5020-5028).Likewise, lymphocytic cells migrating into tumors, such as NK and Tcells, can be suppressed by factors secreted by tumors as well as byinteractions with receptors expressed on the surface of tumor cells thatactivate apoptosis of the immune cells (see, for example, Villunger, etal, 1997, BLOOD 90:12-20). The exposure of these lymphocytes toimmunosuppressive monocytic cells within the tumor bed can furtherreduce their ability to mount an effective anti-tumor response.

Efforts made to overcome the immune suppressive effects of the localtumor microenvironment include targeted immune stimulation, such astreatment with tumor-specific antibody-cytokine fusion proteins.Effective treatment with this approach has been demonstrated in severalmouse tumor metastasis models, however, treatment is far less effectiveas the size of the tumors increases. This is likely due to the increasedlevel of suppressive factors secreted by the tumor mass as well as otherfactors, such as the increase in tumor interstitial fluid pressure(Griffon-Etienne et al. 1999, CANCER RES. 59:3776-3782), a barrier topenetration of solid tumors by therapeutic agents.

While most cancer patients are still treated with one or more courses ofchemotherapy, it is well known that cytotoxic therapy of cancer isdamaging to the immune system. Immune cells are among the most rapidlydividing cells in the human body, and any treatment that kills dividingcells will also kill immune cells. Thus, treatments including radiation,DNA-damaging chemicals, inhibitors of DNA synthesis, and inhibitors ofmicrotubule function all cause damage to the immune system. Bone marrowtransplants are needed as an adjunct to cancer therapy precisely becausethe immune system becomes damaged and needs to be replenished.Methotrexate and other anti-cancer drugs are often used asimmunosuppressants. There is also evidence that anti-cancer treatmentscan specifically inhibit T cell function. For example, patients who havebeen treated for Hodgkin's disease with whole-body irradiation sufferfrom an apparently permanent loss of naïve T cells (Watanabe et al.,1997, Blood 90:3662).

Based on current knowledge it would appear unlikely that standardtreatments (chemotherapy and radiation) and local immune stimulationwould be a useful combination approach for effective treatment ofcancer. Therefore, there is a need in the art for methods that enhanceantibody-cytokine fusion protein mediated immune responses againstpre-selected cell types, for example, tumor cells, and compositionsemployed in such methods.

SUMMARY OF THE INVENTION

It has been discovered that when an antibody-cytokine fusion protein(immunocytokine) is administered to a mammal bearing a tumor or tumormetastases, it is possible to create a more potent anti-tumor responseif it is administered before, simultaneously with, or after treatment ofthe mammal with an immunocytokine uptake enhancing agent that increasesor enhances the therapeutic effect of the antibody-cytokine fusionprotein by enhancing or increasing its uptake by the tumor. It has beenfound that useful immunocytokine uptake enhancing agents comprisealkylating chemotherapeutic agents and taxanes such as paclitaxel. Inparticular, it has been found that such combinations are useful inmediating the immune destruction of the pre-selected cell type, such astumor cells or virus-infected cells.

In one aspect, the invention provides a method of inducing a cytocidalimmune response against a preselected cell-type in a mammal. The methodcomprises administering to the mammal (i) an immunocytokine comprisingan antibody binding site capable of binding the preselected cell-typeand a cytokine capable of inducing such an immune response against thepreselected cell-type, and (ii) an immunocytokine uptake enhancing agentin an amount sufficient to enhance the immune response relative to theimmune response stimulated by the immunocytokine alone.

In a preferred embodiment, the preselected cell-type can be a cancercell present, for example, in a solid tumor, more preferably in alarger, solid tumor (i.e., greater than about 100 mm³). Alternatively,the preselected cell-type can be a cancer cell present in the form ofsmall metastases.

In another preferred embodiment, the immunocytokine uptake enhancingagent can be administered simultaneously with the immunocytokine.Alternatively, the immunocytokine uptake enhancing agent can beadministered prior to administration of the immunocytokine. Furthermore,it is contemplated that the immunocytokine can be administered togetherwith a plurality of different immunocytokine uptake enhancing agents.Alternatively, it is contemplated that an immunocytokine uptakeenhancing agent can be administered together with a plurality ofdifferent immunocytokines.

In another aspect, the invention provides a composition for inducing acytocidal immune response against a preselected cell-type in a mammal.The composition comprises in combination: (i) an immunocytokinecomprising an antibody binding site capable of binding the preselectedcell-type, and a cytokine capable of inducing such an immune responseagainst the preselected cell-type in the mammal, and (ii) animmunocytokine uptake enhancing agent in an amount sufficient to enhancethe cytocidal response induced by the immunocytokine of the combinationrelative to the cytocidal response stimulated by the immunocytokinealone.

In a preferred embodiment, the antibody binding site of theimmunocytokine preferably comprises an immunoglobulin heavy chain or anantigen binding fragment thereof. The immunoglobulin heavy chainpreferably comprises, in an amino-terminal to carboxy-terminaldirection, an immunoglobulin variable (V_(H)) region domain capable ofbinding a preselected antigen, an immunoglobulin constant heavy 1 (CH1)domain, an immunoglobulin constant heavy 2 (CH2) domain, and optionallymay further include an immunoglobulin constant heavy 3 (CH3) domain. Ina more preferred embodiment, the immunocytokine is a fusion proteincomprising an immunoglobulin heavy chain or an antigen binding fragmentthereof fused via a polypeptide bond to the cytokine. Accordingly, apreferred antibody-cytokine fusion protein comprises, in anamino-terminal to carboxy-terminal direction, (i) the antibody bindingsite comprising an immunoglobulin variable region capable of binding acell surface antigen on the preselected cell-type, an immunoglobulin CH1domain, an immunoglobulin CH2 domain (optionally a CH3 domain), and (ii)the cytokine. Methods for making and using such fusion proteins aredescribed in detail in Gillies et al. (1992) Proc. Natl. Acad. Sci. USA89: 1428-1432; Gillies et al. (1998) J. Immunol. 160: 6195-6203; andU.S. Pat. No. 5,650,150.

The immunoglobulin constant region domains (i.e., the CH1, CH2 and/orCH3 domains) may be the constant region domains normally associated withthe variable region domain in a naturally occurring antibody.Alternatively, one or more of the immunoglobulin constant region domainsmay be derived from antibodies different from the antibody used as asource of the variable region domain. In other words, the immunoglobulinvariable and constant region domains may be derived from differentantibodies, for example, antibodies derived from different species. See,for example, U.S. Pat. No. 4,816,567. Furthermore, the immunoglobulinvariable regions may comprise framework region (FR) sequences derivedfrom one species, for example, a human, and complementarity determiningregion (CDR) sequences interposed between the FRs, derived from asecond, different species, for example, a mouse. Methods for making andusing such chimeric immunoglobulin variable regions are disclosed, forexample, in U.S. Pat. Nos. 5,225,539 and 5,585,089.

The antibody-based immunocytokines preferably further comprise animmunoglobulin light chain which preferably is covalently bonded to theimmunoglobulin heavy chain by means of, for example, a disulfide bond.The variable regions of the linked immunoglobulin heavy and light chainstogether define a single and complete binding site for binding thepreselected antigen. In other embodiments, the immunocytokines comprisetwo chimeric chains, each comprising at least a portion of animmunoglobulin heavy chain fused to a cytokine. The two chimeric chainspreferably are covalently linked together by, for example, one or moreinterchain disulfide bonds.

The invention thus provides fusion proteins in which the antigen-bindingspecificity and activity of an antibody is combined with the potentbiological activity of a cytokine. A fusion protein of the presentinvention can be used to deliver the cytokine selectively to a targetcell in vivo so that the cytokine can exert a localized biologicaleffect in the vicinity of the target cell. In a preferred embodiment,the antibody component of the fusion protein specifically binds anantigen on or within a cancer cell and, as a result, the fusion proteinexerts localized anti-cancer activity. In an alternative preferredembodiment, the antibody component of the fusion protein specificallybinds a virus-infected cell, such as an HIV-infected cell, and, as aresult, the fusion protein exerts localized anti-viral activity.

Cytokines that can be incorporated into the immunocytokines of theinvention include, for example, tumor necrosis factors, interleukins,colony stimulating factors, and lymphokines, as well as others known inthe art. Preferred tumor necrosis factors include, for example, tissuenecrosis factor α (TNFα). Preferred interleukins include, for example,interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-5 (IL-5),interleukin-7 (IL-7), interleukin-12 (IL-12), interleukin-15 (IL-15) andinterleukin-18 (IL-18). Preferred colony stimulating factors include,for example, granulocyte-macrophage colony stimulating factor (GM-CSF)and macrophage colony stimulation factor (M-CSF). Preferred lymphokinesinclude, for example, lymphotoxin (LT). Other useful cytokines includeinterferons, including IFN-α, IFN-β and IFN-γ, all of which haveimmunological effects, as well as anti-angiogenic effects, that areindependent of their anti-viral activities.

It has been found that several types of chemotherapeutic agents areeffective immunocytokine uptake enhancing agents. In particular, usefulimmunocytokine uptake enhancing agents include taxanes and alkylatingchemotherapeutic agents. Several taxanes are known in the art (seeBissery and Lavelle, 1997, in Cancer Therapeutics: Experimental andClinical Agents, Chapter 8, B. Teicher, ed.). In a preferred embodiment,the taxane is Taxol, also known as paclitaxel. Other embodiments includethe semisynthetic taxane, docetaxel, which in some tumor models andclinical indications is more efficacious than paclitaxel. Furtherembodiments include additional taxane derivatives, such as those derivedfrom the natural starting material, 10-deacetyl Baccatin III, extractedfrom the needles of the European Yew tree. One such example is theorally available compound, IDN5109, which is also a poor substrate forP-glycoprotein and generally more active against multidrug resistanttumors. In addition to being orally bioavailable, it also has a highertolerated dose and exhibits less neurotoxic side effects (Polizzi etal., 1999, Cancer Res. 59:1036-1040).

Also provided are preferred dosages and administration regimes foradministering the immunocytokines in combination with the immunocytokineuptake enhancing agents.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of a cytokine.

FIG. 2 shows the effect of paclitaxel and an immunocytokine on LLC/KSAtumor volume over time.

FIG. 3 shows the effect of multiple doses of paclitaxel and animmunocytokine on mean tumor volume of time.

FIG. 4 shows the effect of paclitaxel and an immunocytokine on tumorweight in a lung metastasis assay.

FIG. 5 shows the effect of paclitaxel and an immunocytokine on CT26/KSAtumor volume over time.

FIG. 6 shows the effect of paclitaxel and an immunocytokine on tumorweight in a liver metastasis assay.

FIGS. 7A and 7B show the effect of paclitaxel on immunocytokine uptakeby a tumor.

FIG. 8 shows the effect of cyclophosphamide on immunocytokine uptake bya tumor.

FIG. 9 shows the effect of cyclophosphamide and an immunocytokine ontumor weight in a lung metastasis assay.

FIG. 9B shows the effect of cyclophosphamide and an immunocytokine ontumor volume in tumor growth assay.

FIG. 9C shows the effect of cyclophosphamide and an immunocytokine ontumor volume in tumor growth assay.

FIG. 10 shows the effect of carboplatin and an immunocytokine on tumorvolume in a tumor growth assay.

DETAILED DESCRIPTION OF THE INVENTION

Studies have shown that large, solid tumors are much more refractory toantibody-mediated therapeutic intervention, and to immune therapies ingeneral than are disseminated metastatic foci (Sulitzeanu et al. (1993)Adv. Cancer Res. 60: 247-267). It is believed that low responsiveness toantibody-based therapies is based, in part, upon the production ofimmunosuppressive factors by the tumors.

Although the mechanism for tumor eradication is not completelyunderstood, it is contemplated that cytotoxic T lymphocyte (CTL)responses can lead to destruction of cancer cells and provide immunememory. Furthermore, it is contemplated that under certain circumstancesnatural killer (NK) cells are responsible for tumor eradication in theabsence of CTLs. The different immune responses may result from the factthat certain tumors produce different types or amounts of substancescapable of down-regulating T cells. This is especially true for solidtumors, rather than micrometastatic foci, that have reached a criticalmass and are capable of producing and secreting immunosuppressivefactors at levels sufficient to modulate an immune response against thetumors.

It has now been discovered that cytocidal immune responses initiated byan immunocytokine against a preselected cell-type can be enhancedsignificantly by administering the immunocytokine together with animmunocytokine uptake enhancing agent. The combined therapy isparticularly effective in mediating the immune destruction of a diseasedtissue, such as, an established tumor. Without wishing to be bound bytheory, it is contemplated that the immunocytokine uptake enhancingagent increases the penetration of the immunocytokine into the tumormicroenvironment thus making it capable of overcoming the immunesuppressive effect and more effective at activating cellular immuneresponses against the tumor. Similarly, it is contemplated that such amethod may be useful for the treatment of certain viral diseases where asimilar immune suppressive mechanism prevents effective cellularimmunity, for example, in HIV infection. It is contemplated that theimmunocytokine uptake enhancing agent acts synergistically with theimmunocytokine to mediate the immune destruction of a diseased tissuesuch as an established tumor or virally-infected cells. The presentinvention also describes methods for making and using usefulimmunocytokines, as well as assays useful for testing theirpharmacokinetic activities in pre-clinical in vivo animal models whencombined with suitable immunocytokine uptake enhancing agents.

As used herein, the term “immunocytokine uptake enhancing agent” isunderstood to mean any agent that enhances a cytocidal immune responseinduced by an immunocytokine against a pre-selected cell type. Morespecifically, a preferred immunocytokine uptake enhancing agent is atumor uptake enhancing agent that increases the penetration of animmunocytokine into a tumor. Examples of immunocytokine uptake enhancingagents include, but are not limited to, chemotherapeutic agents such astaxanes, DNA damaging agents including alkylating chemotherapeuticagents, radiation therapy agents, and agents that modulate bloodpressure. Preferred taxanes are taxol, docetaxel, 10-deacetyl BaccatinIII, and derivatives thereof. Preferred alkylating agents arecyclophosphamide, carboplatin, cisplatin, and derivatives thereof. Apreferred form of radiation is gamma irradiation. A preferred bloodpressure modulating agent is an angiotensin II agonist, such asangiotensin II itself, preferably administered periodically according tothe general principles described by Netti et al. (Cancer Research [1995]55:5451-8) and Netti et al (Proc. Nat. Acad. Sci. [1999] 96:3137-3142).Immune response may be determined by methods known to one of ordinaryskill in the art and/or as described herein.

As used herein, the term “cytocidal immune response” is understood tomean any immune response in a mammal, either humoral or cellular innature, that is stimulated by an immunocytokine and which either killsor otherwise reduces the viability of a preselected cell-type in themammal. The immune response may include one or more cell types,including T cells, NK cells and macrophages.

As used herein, the term “immunocytokine” is understood to mean a fusionof (i) an antibody binding site having binding specificity for, andcapable of binding a pre-selected antigen, for example, a cell-typespecific antigen, and (ii) a cytokine that is capable of inducing orstimulating a cytocidal immune response typically against a cancer orvirally-infected cell. Examples of pre-selected antigens include cellsurface antigens such as on cancer cells or virally-infected cells, andinsoluble intracellular antigens, for example, of necrotic cells, whichcan remain attached to the cell membrane. Preferred antigens are targetantigens that are characteristic of tumor cells, such as tumor specificantigens. Accordingly, the immunocytokine is capable of selectivelydelivering the cytokine to a target (which typically is a cell) in vivoso that the cytokine can mediate a localized immune response against atarget cell. For example, if the antibody component of theimmunocytokine selectively binds an antigen on a cancer cell, such as acancer cell in a solid tumor, and in particular a larger solid tumor ofgreater than about 100 mm³, the immunocytokine exerts localizedanti-cancer activity. Alternatively, if the antibody component of theimmunocytokine selectively binds an antigen on a virally-infected cell,such as a HIV infected cell, the immunocytokine exerts localizedanti-viral activity.

As used herein, the term “antibody binding site” is understood to meanat least a portion of an immunoglobulin heavy chain, for example, animmunoglobulin variable region capable of binding a pre-selected antigensuch as a cell type. The antibody binding site also preferably comprisesat least a portion of an immunoglobulin constant region including, forexample, a CH1 domain, a CH2 domain, and optionally, a CH3 domain, or atleast a CH2 domain, or one or more portions thereof. Furthermore, theimmunoglobulin heavy chain may be associated, either covalently ornon-covalently, to an immunoglobulin light chain comprising, forexample, an immunoglobulin light chain variable region and optionallylight chain constant region. Accordingly, it is contemplated that theantibody binding site may comprise an intact antibody or a fragmentthereof, or a single chain antibody, capable of binding the preselectedantigen.

With regard to the immunocytokine, it is contemplated that the antibodyfragment may be linked to the cytokine by a variety of ways well knownto those of ordinary skill in the art. For example, the antibody bindingsite preferably is linked via a polypeptide bond or linker to thecytokine in a fusion protein construct. Alternatively, the antibodybinding site may be chemically coupled to the cytokine via reactivegroups, for example, sulfhydryl groups, within amino acid sidechainspresent within the antibody binding site and the cytokine.

As used herein, the term “cytokine” is understood to mean any protein orpeptide, analog or functional fragment thereof, which is capable ofstimulating or inducing a cytocidal immune response against apreselected cell-type, for example, a cancer cell or a virally-infectedcell, in a mammal. Accordingly, it is contemplated that a variety ofcytokines can be incorporated into the immunocytokines of the invention.Useful cytokines include, for example, tumor necrosis factors (TNFs),interleukins (ILs), lymphokines (Ls), colony stimulating factors (CSFs),interferons (IFNs) including species variants, truncated analogs thereofwhich are capable of stimulating or inducing such cytocidal immuneresponses. Useful tumor necrosis factors include, for example, TNF α.Useful lymphokines include, for example, LT. Useful colony stimulatingfactors include, for example, GM-CSF and M-CSF. Useful interleukinsinclude, for example, IL-2, IL-4, IL-5, IL-7, IL-12, IL-15 and IL-18.Useful interferons, include, for example, IFN-α, IFN-β and IFN-γ.

The gene encoding a particular cytokine of interest can be cloned denovo, obtained from an available source, or synthesized by standard DNAsynthesis from a known nucleotide sequence. For example, the DNAsequence of LT is known (see, for example, Nedwin et al. (1985) NUCLEICACIDS RES. 13: 6361), as are the sequences for IL-2 (see, for example,Taniguchi et al. (1983) NATURE 302: 305-318), GM-CSF (see, for example,Gasson et al. (1984) SCIENCE 266: 1339-1342), and TNF α (see, forexample, Nedwin et al. (1985) NUCLEIC A CIDS R ES. 13: 6361).

In a preferred embodiment, the immunocytokines are recombinant fusionproteins produced by conventional recombinant DNA methodologies, i.e.,by forming a nucleic acid construct encoding the chimericimmunocytokine. The construction of recombinant antibody-cytokine fusionproteins has been described in the prior art. See, for example, Gillieset al. (1992) Proc. Natl. Acad. Sci. USA 89: 1428-1432; Gillies et al.(1998) J. Immunol. 160: 6195-6203; and U.S. Pat. No. 5,650,150.Preferably, a gene construct encoding the immunocytokine of theinvention includes, in 5′ to 3′ orientation, a DNA segment encoding animmunoglobulin heavy chain variable region domain, a DNA segmentencoding an immunoglobulin heavy chain constant region, and a DNAencoding the cytokine. The fused gene is assembled in or inserted intoan expression vector for transfection into an appropriate recipient cellwhere the fused gene is expressed. The hybrid polypeptide chainpreferably is combined with an immunoglobulin light chain such that theimmunoglobulin variable region of the heavy chain (V_(H)) and theimmunoglobulin variable region of the light chain (V_(L)) combine toproduce a single and complete site for binding a preselected antigen. Ina preferred embodiment, the immunoglobulin heavy and light chains arecovalently coupled, for example, by means of an interchain disulfidebond. Furthermore, two immunoglobulin heavy chains, either one or bothof which are fused to a cytokine, can be covalently coupled, forexample, by means of one or more interchain disulfide bonds.

Accordingly, methods of the invention are useful to enhance theanti-tumor activity of an immunocytokine used in a therapeutic method totreat a tumor, including immunocytokine compositions and methodsdisclosed in WO99/29732, WO99/43713, WO99/52562, WO99/53958, andWO01/10912, and antibody-based fusion proteins with an altered aminoacid sequence in the junction region. In one embodiment, methods of theinvention are useful in combination with Fc fusion proteins such asFc-interferon-α.

FIG. 1 shows a schematic representation of an exemplary immunocytokine1. In this embodiment, cytokine molecules 2 and 4 are peptide bonded tothe carboxy termini 6 and 8 of CH3 regions 10 and 12 of antibody heavychains 14 and 16. V_(L) regions 26 and 28 are shown paired with V_(H)regions 18 and 20 in a typical IgG configuration, thereby providing twoantigen binding sites 30 and 32 at the amino terminal ends ofimmunocytokine 1 and two cytokine receptor-binding sites 40 and 42 atthe carboxy ends of immunocytokine 1. Of course, in their broaderaspects, the immunocytokines need not be paired as illustrated or onlyone of the two immunoglobulin heavy chains need be fused to a cytokinemolecule.

Immunocytokines of the invention may be considered chimeric by virtue oftwo aspects of their structure. First, the immunocytokine is chimeric inthat it includes an immunoglobulin heavy chain having antigen bindingspecificity linked to a given cytokine. Second, an immunocytokine of theinvention may be chimeric in the sense that it includes animmunoglobulin variable region (V) and an immunoglobulin constant region(C), both of which are derived from different antibodies such that theresulting protein is a V/C chimera. For example, the variable andconstant regions may be derived from naturally occurring antibodymolecules isolatable from different species. See, for example, U.S. Pat.No. 4,816,567. Also embraced are constructs in which either or both ofthe immunoglobulin variable regions comprise framework region (FR)sequences and complementarity determining region (CDR) sequences derivedfrom different species. Such constructs are disclosed, for example, inJones et al. (1986) Nature 321: 522-525, Verhoyen et al. (1988) SCIENCE239: 1534-1535, and U.S. Pat. Nos. 5,225,539 and 5,585,089. Furthermore,it is contemplated that the variable region sequences may be derived byscreening libraries, for example, phage display libraries, for variableregion sequences that bind a preselected antigen with a desiredaffinity. Methods for making and screening phage display libraries aredisclosed, for example, in Huse et al. (1989) Science 246: 1275-1281 andKang et al. (1991) Proc. Natl. Acad. Sci. USA 88: 11120-11123.

The immunoglobulin heavy chain constant region domains of theimmunocytokines can be selected from any of the five immunoglobulinclasses referred to as IgA (Igα), IgD (Igδ), IgE (Igε), IgG (Igγ), andIgM (Igμ). However, immunoglobulin heavy chain constant regions from theIgG class are preferred. Furthermore, it is contemplated that theimmunoglobulin heavy chains may be derived from any of the IgG antibodysubclasses referred to in the art as IgG1, IgG2, IgG3 and IgG4. As isknown, each immunoglobulin heavy chain constant region comprises four orfive domains. The domains are named sequentially as follows:CH1-hinge-CH2—CH3-(—CH4). CH4 is present in IgM, which has no hingeregion. The DNA sequences of the heavy chain domains have cross homologyamong the immunoglobulin classes, for example, the CH2 domain of IgG ishomologous to the CH2 domain of IgA and IgD, and to the CH3 domain ofIgM and IgE. The immunoglobulin light chains can have either a kappa (κ)or lambda (λ) constant chain. Sequences and sequence alignments of theseimmunoglobulin regions are well known in the art (see, for example,Kabat et al., “Sequences of Proteins of Immunological Interest,” U.S.Department of Health and Human Services, third edition 1983, fourthedition 1987, and Huck et al. (1986) NUC. ACIDS RES. 14: 1779-1789).

In preferred embodiments, the variable region is derived from anantibody specific for a preselected cell surface antigen (an antigenassociated with a diseased cell such as a cancer cell orvirally-infected cell), and the constant region includes CH1, and CH2(and optionally CH3) domains from an antibody that is the same ordifferent from the antibody that is the source of the variable region.In the practice of this invention, the antibody portion of theimmunocytokine preferably is non-immunogenic or is weakly immunogenic inthe intended recipient. Accordingly, the antibody portion, as much aspossible, preferably is derived from the same species as the intendedrecipient. For example, if the immunocytokine is to be administered tohumans, the constant region domains preferably are of human origin. See,for example, U.S. Pat. No. 4,816,567. Furthermore, when theimmunoglobulin variable region is derived from a species other than theintended recipient, for example, when the variable region sequences areof murine origin and the intended recipient is a human, then thevariable region preferably comprises human FR sequences with murine CDRsequences interposed between the FR sequences to produce a chimericvariable region that has binding specificity for a preselected antigenbut yet while minimizing immunoreactivity in the intended host. Thedesign and synthesis of such chimeric variable regions are disclosed inJones et al. (1986) Nature 321: 522-525, Verhoyen et al. (1988) SCIENCE239: 1534-1535, and U.S. Pat. Nos. 5,225,539 and 5,585,089. The cloningand expression of a humanized antibody-cytokine fusion protein, KS-1/4anti-EpCAM antibody-IL-12 fusion protein, as well as its ability toeradicate established colon carcinoma metastases has been described inGillies et al. (1998) J. Immunol. 160: 6195-6203.

The gene encoding the cytokine is joined, either directly or by means ofa linker, for example, by means of DNA encoding a (Gly₄-Ser)₃ linker inframe to the 3′ end of the gene encoding the immunoglobulin constantregion (e.g., a CH2 or CH3 exon). In certain embodiments, the linker cancomprise a nucleotide sequence encoding a proteolytic cleavage site.This site, when interposed between the immunoglobulin constant regionand the cytokine, can be designed to provide for proteolytic release ofthe cytokine at the target site. For example, it is well known thatplasmin and trypsin cleave after lysine and arginine residues at sitesthat are accessible to the proteases. Many other site-specificendoproteases and the amino acid sequences they cleave are well-known inthe art. Preferred proteolytic cleavage sites and proteolytic enzymesthat are reactive with such cleavage sites are disclosed in U.S. Pat.Nos. 5,541,087 and 5,726,044.

The nucleic acid construct optionally can include the endogenouspromoter and enhancer for the variable region-encoding gene to regulateexpression of the chimeric immunoglobulin chain. For example, thevariable region encoding genes can be obtained as DNA fragmentscomprising the leader peptide, the VJ gene (functionally rearrangedvariable (V) regions with joining (J) segment) for the light chain, orVDJ gene for the heavy chain, and the endogenous promoter and enhancerfor these genes. Alternatively, the gene encoding the variable regioncan be obtained apart from endogenous regulatory elements and used in anexpression vector which provides these elements.

Variable region genes can be obtained by standard DNA cloning proceduresfrom cells that produce the desired antibody. Screening of the genomiclibrary for a specific functionally rearranged variable region can beaccomplished with the use of appropriate DNA probes such as DNA segmentscontaining the J region DNA sequence and sequences downstream.Identification and confirmation of correct clones is achieved bysequencing the cloned genes and comparison of the sequence to thecorresponding sequence of the full length, properly spliced mRNA.

The target antigen can be a cell surface antigen of a tumor or cancercell, a virus-infected cell or another diseased cell. The target antigenmay also be an insoluble intracellular antigen of a necrotic cell. (see,for example, U.S. Pat. No. 5,019,368) Genes encoding appropriatevariable regions can be obtained generally from immunoglobulin-producinglymphoid cell lines, For example, hybridoma cell lines producingimmunoglobulin specific for tumor associated antigens or viral antigenscan be produced by standard somatic cell hybridization techniques wellknown in the art (see, for example. U.S. Pat. No. 4,196,265). Theseimmunoglobulin producing cell lines provide the source of variableregion genes in functionally rearranged form. The variable region genestypically will be of murine origin because this murine system lendsitself to the production of a wide variety of immunoglobulins of desiredspecificity. Furthermore, variable region sequences may be derived byscreening libraries, for example, phage display libraries, for variableregion sequences that bind a preselected antigen with a desiredaffinity. Methods for making and screening phage display libraries aredisclosed, for example, in Huse et al. (1989) Science 246: 1275-1281 andKang et al. (1991) Proc. Natl. Acad. Sci. USA 88: 11120-11123.

The DNA fragment encoding the functionally active variable region geneis linked to a DNA fragment containing the gene encoding the desiredconstant region (or a portion thereof). Immunoglobulin constant regions(heavy and light chain) can be obtained from antibody-producing cells bystandard gene cloning techniques. Genes for the two classes of humanlight chains (κ and λ) and the five classes of human heavy chains (α, δ,ε, γ and μ) have been cloned, and thus, constant regions of human originare readily available from these clones.

The fused gene encoding the hybrid immunoglobulin heavy chain isassembled or inserted into an expression vector for incorporation into arecipient cell. The introduction of the gene construct into plasmidvectors can be accomplished by standard gene splicing procedures. Thechimeric immunoglobulin heavy chain can be co-expressed in the same cellwith a corresponding immunoglobulin light chain so that a completeimmunoglobulin can be expressed and assembled simultaneously. For thispurpose, the heavy and light chain constructs can be placed in the sameor separate vectors.

Recipient cell lines are generally lymphoid cells. The preferredrecipient cell is a myeloma (or hybridoma). Myelomas can synthesize,assemble, and secrete immunoglobulins encoded by transfected genes andthey can glycosylate proteins. Particularly preferred recipient or hostcells include Sp2/0 myeloma which normally does not produce endogenousimmunoglobulin, and mouse myeloma NS/0 cells. When transfected, the cellproduces only immunoglobulin encoded by the transfected gene constructs.Transfected myelomas can be grown in culture or in the peritoneum ofmice where secreted immunocytokine can be recovered from ascites fluid.Other lymphoid cells such as B lymphocytes can be used as recipientcells.

There are several methods for transfecting lymphoid cells with vectorscontaining the nucleic acid constructs encoding the chimericimmunoglobulin chain. For example, vectors may be introduced intolymphoid cells by spheroblast fusion (see, for example, Gillies et al.(1989) BIOTECHNOL. 7: 798-804). Other useful methods includeelectroporation or calcium phosphate precipitation (see, for example,Sambrook et al. eds (1989) “Molecular Cloning: A Laboratory Manual,”Cold Spring Harbor Press).

Other useful methods of producing the immunocytokines include thepreparation of an RNA sequence encoding the construct and itstranslation in an appropriate in vivo or in vitro expression system. Itis contemplated that the recombinant DNA methodologies for synthesizinggenes encoding antibody-cytokine fusion proteins, for introducing thegenes into host cells, for expressing the genes in the host, and forharvesting the resulting fusion protein are well known and thoroughlydocumented in the art. Specific protocols are described, for example, inSambrook et al. eds (1989) “Molecular Cloning: A Laboratory Manual,”Cold Spring Harbor Press.

It is understood that the chemically coupled immunocytokines may beproduced using a variety of methods well known to those skilled in theart. For example, the antibody or an antibody fragment may be chemicallycoupled to the cytokine using chemically reactive amino acid side chainsin the antibody or antibody fragment and the cytokine. The amino acidside chains may be covalently linked, for example, via disulfide bonds,or by means of homo- or hetero-bifunctional crosslinking reagentsincluding, for example, N-succinimidyl 3(-2-pyridyylditio)propionate,m-maleimidobenzoyl-N-hydroxysuccinate ester,m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester, and1,4-di-[3′(2′-pyridylthio)propionamido]butane, all of which areavailable commercially from Pierce, Rockford, Ill.

According to methods of the invention, the combination ofimmunocytokines with immunocytokine uptake enhancing agents is usefulfor enhanced stimulation of the immune system, thereby resulting in acytotoxic response at the site of the targeted cell type, for example,tumor or other disease cells. A combination of an immunocytokine and animmunocytokine uptake enhancing agent would be expected to have nocombined or synergistic anti-tumor effect in vitro since theimmunocytokine alone is non-cytotoxic.

Without wishing to be bound by any particular theory, it is believedthat the effects of combined therapy in vivo may include enhanced uptakeof one of the agents by the action of the other resulting in either orboth (1) increased chemotherapeutic cytotoxicity (if the immunocytokineincreased the uptake of the chemotherapeutic immunocytokine uptakeenhancing agent into tumor cells); and/or (2) increased immunestimulation (if the immunocytokine uptake enhancing agent in some wayincreased uptake of the immunocytokine into the tumor). With respect tomechanism (1), earlier studies have shown that it is possible toincrease the uptake of radiolabeled antibodies (and presumably, smallmolecule drugs) into tumors by prior treatment with high doses of anantibody-IL2 immunoconjugate that induces a local vascular leak (see forexample, Hornick et al., 1999, CLIN CANCER RES 5:51-60). If thisparticular mechanism is operative in the combination therapy ofimmunocytokines and immunocytokine uptake enhancing agents, it would benecessary to first treat the tumor-bearing animal with theimmunocytokine. However, if a single dose of an immunocytokine uptakeenhancing agent given prior to treatment with an immunocytokine resultedin a synergistic effect on anti-tumor activity, then such a mechanismcould not be operative. Rather, a more likely explanation would be thattreatment with an immunocytokine uptake enhancing agent increased theuptake of the immunocytokine by mechanism (2). This hypothesis could befurther supported by demonstrating that co-administration with animmunocytokine uptake enhancing agent increases the uptake of aradiolabeled immunocytokine into a solid tumor.

According to methods of the invention, an advantage of the combinationtherapy is that the administration of an immunocytokine enhances thecytotoxic effect of a chemotherapeutic agent that acts as immunocytokineuptake enhancing agent. Therefore, a lower dosage of thechemotherapeutic agent may be administered to a patient. Accordingly,the suppression of some aspects of a patient's immune system, oftenassociated with treatment using a chemotherapeutic agent, is reduced. Inone embodiment of the invention, a single dose of chemotherapeuticimmunocytokine uptake enhancing agent is administered to a patientbefore an immunocytokine is administered. The chemotherapeuticimmunocytokine uptake enhancing agent is administered preferably betweenabout 4 days and about 4 hours, and most preferably about 24-48 hours,before the immunocytokine. In another embodiment of the inventionseveral doses of the chemotherapeutic immunocytokine uptake enhancingagent are administered to a patient before the immunocytokine isadministered. In further embodiments of the invention, thechemotherapeutic immunocytokine uptake enhancing agent may beadministered before, at the same time, and/or after the immunocytokine.

Paclitaxel is an example of a chemotherapeutic immunocytokine uptakeenhancing agent that can suppress or compromise aspects of a patient'simmune system. While most immune potentiating effects of paclitaxel aremediated through macrophage/monocyte cells, many studies on lymphocytefunction indicate a detrimental effect of paclitaxel on this subset. Forexample, paclitaxel treatment was found to severely compromise theproliferative capacity of lymphocytes in both normal and tumor-bearingmice (Mullins et al., 1998, IMMUNOPHARMACOL IMMUNOTOXICOL 20:473-492),and to impair both the cytotoxicity of NK cells and the generation oflymphokine-activated cytotoxicity in cell cultures containing IL-2(Chuang et al., 1993, GYNECOL ONCOL 49:291-298). In fact, the availableevidence points to the lymphocyte subset of cells as the essentialeffector population in the anti-tumor activity of immunocytokines (Lodeet al, 1998, PHARMACOL THER 80:277-292. Experimental evidence containedwithin the present invention has revealed several novel findings thatwould not have been predicted by the prior art, especially with respectto the order of drug administration.

Taxanes may be co-administered simultaneously with the immunocytokine,or administered separately by different routes of administration.Compositions of the present invention may be administered by any routethat is compatible with the particular molecules. Thus, as appropriate,administration may be oral or parenteral, including intravenous andintraperitoneal routes of administration.

The compositions of the present invention may be provided to an animalby any suitable means, directly (e.g., locally, as by injection,implantation or topical administration to a tissue locus) orsystemically (e.g., parenterally or orally). Where the composition is tobe provided parenterally, such as by intravenous, subcutaneous,ophthalmic, intraperitoneal, intramuscular, buccal, rectal, vaginal,intraorbital, intracerebral, intracranial, intraspinal,intraventricular, intrathecal, intracisternal, intracapsular, intranasalor by aerosol administration, the composition preferably comprises partof an aqueous or physiologically compatible fluid suspension orsolution. Thus, the carrier or vehicle is physiologically acceptable sothat in addition to delivery of the desired composition to the patient,it does not otherwise adversely affect the patient's electrolyte and/orvolume balance. The fluid medium for the agent thus can comprise normalphysiologic saline (e.g., 9.85% aqueous NaCl, 0.15 M, pH 7-7.4). Formany taxanes, the formulations are generally more complex, due to theirgenerally unfavorable solubility properties. For example, the standardformulation for paclitaxel is 10% Cremophor, 10% ethanol, and 80% saline(0.9% NaCl), while the formulation for docetaxel is a 1:1ethanol:polysorbate 80 solution that is diluted 1:10 into 5% glucosesolution prior to administration (Bissery and Lavelle, 1999). However,other formulations including taxanes and newly synthesized analogs willbe recognized and/or routinely developed by those skilled in the art.

Preferred dosages of the immunocytokine per administration are withinthe range of 0.1 mg/m²-100 mg/m², more preferably, 1 mg/m²-20 mg/m², andmost preferably 2 mg/m²-6 mg/m². Preferred dosages of the immunocytokineuptake enhancing agent will depend generally upon the type ofimmunocytokine uptake enhancing agent used, however, optimal dosages maybe determined using routine experimentation. Administration of theimmunocytokine and/or the immunocytokine uptake enhancing agent may beby periodic bolus injections, or by continuous intravenous orintraperitoneal administration from an external reservoir (for example,from an intravenous bag) or internal (for example, from a bioerodableimplant). Furthermore, it is contemplated that the immunocytokine of theinvention may also be administered to the intended recipient togetherwith a plurality of different immunocytokine uptake enhancing agents. Itis contemplated, however, that the optimal combination ofimmunocytokines and immunocytokine uptake enhancing agents, modes ofadministration, dosages may be determined by routine experimentationwell within the level of skill in the art.

A variety of methods can be employed to assess the efficacy of combinedtherapy using antibody-cytokine fusion proteins and immunocytokineuptake enhancing agents on immune responses. For example, the animalmodel described in the examples below, or other suitable animal models,can be used by a skilled artisan to test which immunocytokine uptakeenhancing agents, or combinations of immunocytokine uptake enhancingagents, are most effective in acting synergistically with animmunocytokine (for example, an antibody-IL2 fusion protein) to enhancethe immune destruction of established tumors. The immunocytokine uptakeenhancing agent, or combination of immunocytokine uptake enhancingagents, can be administered prior to, or simultaneously with, the courseof immunocytokine therapy and the effect on the tumor can beconveniently monitored by volumetric measurement. Further, as novelimmunocytokine uptake enhancing agents are identified, a skilled artisanwill be able to use the methods described herein to assess the potentialof these novel compounds to enhance or otherwise modify the anti-canceractivity of antibody-cytokine fusion proteins.

Alternatively, following therapy, tumors can be excised, sectioned andstained via standard histological methods, or via specificimmuno-histological reagents in order to assess the effect of thecombined therapy on the immune response. For example, simple stainingwith hematoxolin and eosin can reveal differences in lymphocyticinfiltration into the solid tumors which is indicative of a cellularimmune response. Furthermore, immunostaining of sections with antibodiesto specific classes of immune cells can reveal the nature of an inducedresponse. For example, antibodies that bind to CD45 (a general leukocytemarker), CD4 and CD8 (for T cell subclass identification), and NK1.1 (amarker on NK cells) can be used to assess the type of immune responsethat has been mediated by the immunocytokines of the invention.

Alternatively, the type of immune response mediated by theimmunocytokines can be assessed by conventional cell subset depletionstudies described, for example, in Lode et al. (1998) Blood 91:1706-1715. Examples of depleting antibodies include those that reactwith T cell markers CD4 and CD8, as well as those that bind the NKmarkers NK1.1 and asialo GM. Briefly, these antibodies are injected tothe mammal prior to initiating antibody-cytokine treatment at fairlyhigh doses (for example, at a dose of about 0.5 mg/mouse), and are givenat weekly intervals thereafter until the completion of the experiment.This technique can identify the cell-types necessary to elicit theobserved immune response in the mammal.

In another approach, the cytotoxic activity of splenocytes isolated fromanimals having been treated with the combination therapy can be comparedwith those from the other treatment groups. Splenocyte cultures areprepared by mechanical mincing of recovered, sterile spleens by standardtechniques found in most immunology laboratory manuals. See, forexample, Coligan et al. (eds) (1988) “Current Protocols in Immunology,”John Wiley & Sons, Inc. The resulting cells then are cultured in asuitable cell culture medium (for example, DMEM from GIBCO) containingserum, antibiotics and a low concentration of IL-2 (˜10 U/mL). Forexample, in order to compare NK activity, 3 days of culture normally isoptimal, whereas, in order to compare T cell cytotoxic activity, 5 daysof culture normally is optimal. Cytotoxic activity can be measured byradioactively labeling tumor target cells (for example, LLC cells) with⁵¹Cr for 30 min. Following removal of excess radiolabel, the labeledcells are mixed with varying concentrations of cultured spleen cells for4 hr. At the end of the incubation, the ⁵¹Cr released from the cells ismeasured by a gamma counter which is then used to quantitate the extentof cell lysis induced by the immune cells. Traditional cytotoxic Tlymphocyte (or CTL) activity is measured in this way.

The invention is illustrated further by the following non-limitingexamples.

Example 1 Animal Models

Murine cancer models were developed to study the effect of combiningimmunocytokines and taxanes in mediating effective cytotoxic responsesagainst a tumor. The immunocytokines used in the following examples bindEpCAM, a human tumor antigen found on most epithelial derived tumors.(see, Perez and Walker (1989) J. Immunol. 142: 3662-3667). In order totest the efficacy in an immuno-competent murine model, it was necessaryto express the human antigen on the surface of a mouse tumor cell thatis syngeneic with the mouse host. Lewis lung carcinoma (LLC) cells, awell known mouse lung cancer cell line, was the first cell line selectedfor this purpose. This cell line is known to produce high levels ofinhibitors of the immune system and to induce IL-10 production fromimmune cells in the tumor microenvironment leading to localized immunesuppression (Sharma et al., 1999, J IMMUNOL 163:5020-5028). The humantumor antigen, EpCAM (also referred to as KSA), was expressed on thesurface of LLC cells so that it could be targeted in vivo withimmunocytokines derived from the mouse anti-EpCAM antibody, KS-1/4. Thiswas accomplished by transducing the EpCAM cDNA sequence with arecombinant retroviral vector as described (Gillies, U.S. patentapplication Ser. No. 09/293,042) resulting in a cell line designatedLLC/KSA. These cells were maintained in DMEM, supplemented with 10% heatinactivated fetal bovine serum, L-glutamine, penicillin/streptomycin andGeneticin (GIBCO) at 37° C. and 7.0% CO₂.

Additional cell lines representing carcinoma of different tissue originswere engineered in a similar manner. 4T1, a non-immunogenic murinemammary carcinoma cell line, was provided by Dr. Paul Sondel (Univ. ofWisconsin). This line grows slowly and progressively after subcutaneousimplantation and spontaneously metastasizes to many organs even prior tosurgical removal of the primary tumor. It is also possible to induceexperimental metastases in the lung by intravenous injection. CT26, amurine colon carcinoma cell line, derived by intrarectal injection ofN-nitroso-N-methylurethane in BALB/C mice, was provided by Dr. I. J.Fidler (MD Anderson Cancer Center, Houston, Tex.). 4T1 and CT26 cellswere transfected with Ep-CAM as described (Gillies et al., 1998, JIMMUNOL 160:6195-6203). 4T1/KSA cells were maintained in RPMI,supplemented with 10% heat inactivated fetal bovine serum, L-glutamine,penicillin/streptomycin and Geneticin (GIBCO) at 37° C. and 7.0% CO₂.CT26/KSA cells were maintained in DMEM, supplemented with 10% heatinactivated fetal bovine serum, L-glutamine, vitamins, sodium pyruvate,non-essential amino acids, penicillin/streptomycin and Geneticin (GIBCO,Gaithersberg, Md.) at 37° C. and 7.0% CO₂. Geneticin was added to thetransfected cells to maintain KSA expression. All of the transfectedcell lines grow progressively as skin tumors (after subcutaneousinjection) or as metastases (after intravenous injection) and kill themice, despite their expression of the human EpCAM molecule (a potentialforeign antigen) on their cell surface.

For tumor growth studies either LLC/KSA or CT26/KSA tumors wereimplanted subcutaneously on the backs of mice. For LLC/KSA studies,tumors were transplanted from several stock tumors that had beeninjected with a single cell suspension of 1×10⁶ cells in 100 ul of PBS.After about two weeks, tumors were aseptically collected, passed througha sieve fitted with a 150 μm screen. Cells were then passed through asyringe and 23 gauge needle tow or three times, washed twice, andresuspended in PBS. A single cell suspension of 1×10⁶ LLC/KSA cells in100 ul of PBS was injected subcutaneously using a 30½ gauge needle onthe backs of mice. For CT26/KSA studies, cells growing exponentially inculture were injected as a single cell suspension of 1×10⁶ cells in 100μl of PBS. After tumors had become established, about 2 weeks afterimplantation, dosing was initiated on Day 0. Tumors were measured withcalipers in three dimensions twice weekly. Tumor volumes were calculatedusing the equation:

Volume=½×4/3π(L/2×W/2×H)

where L=length, W=width and H=height of the tumor. Animals were weighedand general health was monitored during the course of the study. Whentumors became necrotic or if animals became moribund, the animals wereeuthanized by CO₂ asphyxiation.

Data are presented in graphic form. Graphs depict individual or averagetumor volumes (+/−SEM) during and after dosing. Data are also expressedas the percent of control of average tumor volumes from treated micerelative to vehicle treated mice. Student's t test was performed on theindividual tumor volumes to determine significant differences.

For experimental hepatic metastases studies, mice were anesthetizedusing 80 mg/kg ketamine HCL (Fort Dodge Animal Health, Fort Dodge, Iowa)and 5 mg/kg xylazine (Bayer, Shawnee Mission, Kans.). A single cellsuspension of 1×10⁵ CT26/KSA cells in 100 μl of DMEM containing 25 mMHEPES (GIBCO) was injected using a 27½ gauge needle beneath the spleniccapsule over a period of 60 seconds on Day 0. After another 2 minutesthe splenic vessels were cauterized with a cautery unit (Roboz,Rockville, Md.) and the spleen removed. Animals were sutured usingautoclips. Three weeks after inoculation the animals were sacrificed;their livers were removed and weighed. The livers were then fixed andstained in Bouin's solution (Sigma, St. Louis Mo.).

Data are presented in graphic form. Graphs depict average tumor burdens(+/−SEM) at the time of sacrifice. Tumor burdens were determined bysubtracting the weight of a normal liver from the weight of theexperimental livers. Data are also expressed as the percent of controlof the average tumor burden from treated mice relative to vehicletreated mice. Student's t test was performed on the individual tumorburdens to determine significant differences.

For experimental lung metastases studies, a single cell suspension of2.5×10⁵ 4T1/KSA cells in 100 μl of PBS was slowly injected using a 27½gauge needle into the lateral tail vein on Day 0. About 3 weeks-afterinoculation animals were sacrificed; their lungs were removed andweighed. The lungs were then fixed and stained in Bouin's solution(Sigma). Data are presented in graphic form. Graphs depict average tumorburdens (+/−SEM) at time of sacrifice. Tumor burden was determined bysubtracting the weight of a normal lung from the weight of theexperimental lungs. Data are also expressed as the percent of control ofaverage tumor burden from treated mice relative to vehicle treated mice.Student's t test was performed on the individual tumor burdens todetermine significant differences.

Example 2 Preparation of Antibody-Fusion Proteins (Immunocytokines)

Several antibody-cytokine fusion proteins are discussed in the followingexamples.

huKS-huγl-huIL2 (Abbreviated, KS-IL2)

A gene encoding huKS-huγ1-huIL2 fusion protein was prepared andexpressed essentially as described in Gillies et al. (1998) J. Immunol.160: 6195-6203 and U.S. Pat. No. 5,650,150. Briefly, humanized variableregions of the mouse KS1/4 antibody (Varki et al., (1984) Cancer Res.44: 681-687) were modeled using the methods disclosed in Jones et al.(1986) Nature 321: 522-525, which involved the insertion of the CDRs ofeach KS1/4 variable region into the consensus framework sequences of thehuman variable regions with the highest degree of homology. Molecularmodeling with a Silicon Graphics Indigo work station implementing BioSymsoftware confirmed that the shapes of the CDRs were maintained. Theprotein sequences then were reverse translated, and genes constructed bythe ligation of overlapping oligonucleotides.

The resulting variable regions were inserted into an expression vectorcontaining the constant regions of the human κ light chain and the humanCγ1 heavy chain essentially as described in Gillies et al. (1992) Proc.Natl. Acad. Sci. USA 89: 1428-1432, except that the metallothioneinpromoters and immunoglobulin heavy chain enhancers were replaced by theCMV promoter/enhancer for the expression of both chains. Fusions of themature sequences of IL-2 to the carboxy terminus of the human heavychains were prepared as described in Gillies et al. (1992) Proc. Natl.Acad. Sci. USA 89:1428-1432, except that the 3′ untranslated regions ofthe IL-2 gene was derived from the SV40 poly(A) region.

The IL-2 fusion protein was expressed by transfection of the resultingplasmid into NS/0 myeloma cell line with selection medium containing 0.1μM methotrexate (MTX). Briefly, in order to obtain stably transfectedclones, plasmid DNA was introduced into the mouse myeloma NS/0 cells byelectroporation. NS/0 cells were grown in Dulbecco's modified Eagle'smedium supplemented with 10% fetal bovine serum. About 5×10⁶ cells werewashed once with PBS and resuspended in 0.5 mL PBS. Ten μg of linearizedplasmid DNA then was incubated with the cells in a Gene Pulser Cuvette(0.4 cm electrode gap, BioRad) on ice for 10 min. Electroporation wasperformed using a Gene Pulser (BioRad, Hercules, Calif.) with settingsat 0.25 V and 500 μF. Cells were allowed to recover for 10 min. on ice,after which they were resuspended in growth medium and then plated ontotwo 96 well plates. Stably transfected clones were selected by growth inthe presence of 100 nM methotrexate, which was introduced two dayspost-transfection. The cells were fed every 3 days for three more times,and MTX-resistant clones appeared in 2 to 3 weeks.

Expressing clones were identified by Fc or cytokine ELISA using theappropriate antibodies (see, for example, Gillies et al. (1989)Biotechnol. 7: 798-804). The resulting fusion protein was purified bybinding, and elution from protein A Sepharose (Pharmacia), in accordancewith the manufacturer's instructions.

huKS-huγ4-huIL2

A gene encoding the huKS-huγ4-huIL2 fusion protein was constructed andexpressed essentially as described in U.S. Ser. No. 09/256,156, filedFeb. 24, 1999, which claims priority to U.S. Ser. No. 60/075,887, filedFeb. 25, 1998.

Briefly, an Igγ4 version of the huKS-huγ1-huIL2 fusion protein,described above, was prepared by removing the immunoglobulin constantregion Cγ1 gene fragment from the huKS-huγ1-huIL2 expression vector andreplacing it with the corresponding sequence from the human Cγ4 gene.Sequences and sequence alignments of the human heavy chain constantregions Cγ1, Cγ2, Cγ3, and Cγ4 are disclosed in Huck et al. (1986) NUC.ACIDS RES. 14: 1779-1789.

The swapping of the Cγ1 and Cγ4 fragments was accomplished by digestingthe original Cγ1-containing plasmid DNA with Hind III and Xho I andpurifying a large 7.8 kb fragment by agarose gel electrophoresis. Asecond plasmid DNA containing the Cy4 gene was digested with Hind IIIand Nsi I and a 1.75 kb fragment was purified. A third plasmidcontaining the human IL-2 cDNA and SV40 polyA site, fused to thecarboxyl terminus of the human Cγ1 gene, was digested with Xho I and NsiI and the small 470 by fragment was purified. All three fragments wereligated together in roughly equal molar amounts. The ligation productwas used to transform competent E. coli and colonies were selected bygrowth on plates containing ampicillin. Correctly assembled recombinantplasmids were identified by restriction analyses of plasmid DNApreparations from isolated transformants and digestion with Fsp I wasused to discriminate between the Cγ1 (no Fsp I) and Cy4 (one site) geneinserts.

The final vector, containing the Cγ4-IL2 heavy chain replacement, wasintroduced into NS/0 mouse myeloma cells by electroporation (0.25 V and500 μF) and transfectants were selected by growth in medium containingmethotrexate (0.1 μM). Cell clones expressing high levels of thehuKS-huγ4-huIL2 fusion protein were identified, expanded, and the fusionprotein purified from culture supernatants using protein A Sepharosechromatography. The purity and integrity of the Cy4 fusion protein wasdetermined by SDS-polyacrylamide gel electrophoresis. IL-2 activity wasmeasured in a T-cell proliferation assay (Gillis et al. (1978) J.Immunol. 120: 2027-2032) and was found to be identical to that of theγ1-construct.

huKS-muγ2a-muIL2

A gene encoding the huKS-muγ2a-muIL2 fusion protein was constructed byreplacing the human antibody constant regions and human IL-2 of thehuKS-huγ1-huIL2 fusion protein, as described above, with thecorresponding murine sequences. Specifically, the human Cγ1-IL2 DNA wasreplaced with a murine Cγ2a cDNA fragment fused to a DNA encoding murineIL-2. Briefly, the V_(H) region of the huKS was joined in frame to themurine γ2a cDNA by performing overlapping PCR using overlappingoligonucleotide primers:

(SEQ ID NO: 1) (sense) 5′CC GTC TCC TCA GCC AAA ACA ACA GCC CCA TCG GTC; (SEQ ID NO: 2)(antisense) 5′ GG GGC TGT TGT TTT GGC TGA GGA GAC GGT GAC TGA CG;(SEQ ID NO: 3) (sense) 5′ C TTA AGC CAG ATC CAG TTG GTG CAG; and(SEQ ID NO: 4) (antisense) 5′ CC CGG GGT CCG GGA GAA GCT CTT AGT C.

The oligonucleotides of SEQ ID NOS: 1 and 2 were designed to hybridizeto the junction of the V_(H) domain of huKS and the constant region ofmurine γ2a cDNA (in italics). In the first round of PCR, there were twoseparate reactions. In one reaction, the V_(H) of huKS DNA was used asthe template with the oligonucleotides of SEQ ID NOS: 2 and 3. Theprimer of SEQ ID NO: 3 introduced an AflII (CTTAAG) restriction siteupstream of the sequence encoding the mature amino terminus of huKSV_(H) (in bold). In another reaction, murine γ2a cDNA was used as thetemplate with the oligonucleotides SEQ ID NOS: 1 and 4. The primer ofSEQ ID NO: 4 hybridized to the cDNA encoding the region around theC-terminus of γ2a and introduced a XmaI (CCCGGG) restriction site forsubsequent ligation to the muIL2 cDNA. PCR products from the tworeactions were mixed and subjected to a second round of PCR, using theoligonucleotides of SEQ ID NOS: 3 and 4. The resulting PCR product wascloned, and upon sequence verification, the AflII-XmaI fragment encodingthe V_(H) of huKS and the murine γ2a constant region was used forligation to the DNA encoding the signal peptide at the AflII site andthe muIL2 cDNA at the XmaI site.

The murine IL2 cDNA was cloned from mRNA of murine peripheral bloodmononuclear cells using the oligonucleotides set forth in SEQ ID NOS: 5and 6, namely:

(SEQ ID NO: 5) (sense) 5′ GGC CCG GGT AAA GCA CCC ACT TCA AGC TCC; and(SEQ ID NO: 6) (antisense) 5′ CCCTCGAGTTATTGAGGGCTTGTTG.

The primer of SEQ ID NO: 5 adapted the muIL2 (sequence in bold) to bejoined to mu γ2a at the XmaI restriction site (CCCGGG). The primer ofSEQ ID NO: 6 introduced an XhoI restriction site (CTCGAG) immediatelyafter the translation termination codon (antisense in bold).

Similarly, the variable light (V_(L)) domain of huKS was joined to themu κ cDNA sequence by overlapping PCR. The overlapping oligonucleotidesused included

(SEQ ID NO: 7) (sense) 5′ G GAA ATA AAA CGG GCT GAT GCT GCA CCA ACT G;(SEQ ID NO: 8) (antisense) 5′GC AGC ATC AGC CCGTT TTA TTT CCA GCT TGG TCC; (SEQ ID NO: 9) (sense) 5′C TTA AGC GAG ATC GTG CTG ACC CAG; and (SEQ ID NO: 10) (antisense) 5′CTC GAG CTA ACA CTC ATT CCT GTT GAA GC.

The oligonucleotides were designed to hybridize to the junction of theV_(L) of huKS and the constant region of murine κ cDNA (in italics). Inthe first round of PCR, there were two separate reactions. In onereaction, the V_(L) of huKS DNA was used as template, with theoligonucleotides set forth in SEQ ID NOS: 8 and 9, which introduced anAflII (CTTAAG) restriction site upstream of the sequence encoding themature amino terminus of huKS V_(L) (in bold). In the other reaction,murine κ cDNA was used as template, with the oligonucleotides set forthin SEQ ID NOS: 7 and 10, which introduced an XhoI restriction site afterthe translation termination codon (antisense in bold).

PCR products from the two reactions were mixed and subjected to a secondround of PCR using the oligonucleotide primers set forth in SEQ ID NOS:9 and 10. The resultant PCR product was cloned, and upon sequenceverification, the AflII-XhoI fragment encoding the V_(L) of huKS and themurine κ constant region was ligated to the DNA encoding the signalpeptide at the AflII site.

Both the murine heavy and light chain sequences were used to replace thehuman sequences in pdHL7. The resulting antibody expression vector,containing a dhfr selectable marker gene, was electroporated (6.25 V,500 μF) into murine NS/0 myeloma cells and clones selected by culturingin medium containing 0.1 μM methotrexate. Transfected clones, resistantto methotrexate, were tested for secretion of antibody determinants bystandard ELISA methods. The fusion proteins were purified via protein ASepharose chromatography according to the manufacturers instructions.

huKS-muγ2a-muIL12

A gene encoding the huKS-muγ2a-muIL12 fusion protein was constructed andexpressed essentially as described in U.S. Ser. No. 08/986,997, filedDec. 8, 1997, and Gillies et al. (1998) J. Immunol. 160: 6195-6203.Briefly, this was accomplished by fusing the murine p35 IL-12 subunitcDNA to the huKS-muγ2a heavy chain coding region prepared previously.The resulting vector then was transfected into an NS/0 myeloma cell linepre-transfected with, and capable of expressing p40 IL-12 subunit. Inother words, a cell line was transfected with p40 alone and a stable,high expressing cell was selected, which was then used as a recipientfor transfection by the p35 containing fusion protein (i.e., sequentialtransfection).

The murine p35 and p40 IL-12 subunits were isolated by PCR from mRNAprepared from spleen cells activated with Concanavalin A (5 μg/mL inculture medium for 3 days). The PCR primers used to isolate the p35encoding nucleic acid sequence which also adapted the p35 cDNA as anXmaI-XhoI restriction fragment included:

5′ CCCCGGGTAGGGTCATTCCAGTCTCTGG; (SEQ ID NO: 11) and 5′CTCGAGTCAGGCGGAGCTCAGATAGC. (SEQ ID NO: 12)

The PCR primer used to isolate the p40 encoding nucleic acid sequenceincluded:

5′ TCTAGACCATGTGTCCTCAGAAGCTAAC; (SEQ ID NO: 13) and 5′CTCGAGCTAGGATCGGACCCTGCAG. (SEQ ID NO: 14)

A plasmid vector (pdHL7-huKS-muγ2a-p35) was constructed as described(Gillies et al. J. Immunol. Methods 125: 191) that contained a dhfrselectable marker gene, a transcription unit encoding a humanized KSantibody light chain, and a transcription unit encoding a murine heavychain fused to the p35 subunit of mouse IL-12. The fusion was achievedby ligation of the XmaI to XhoI fragment of the adapted p35 subunitcDNA, to a unique XmaI site at the end of the CH3 exon of the murine γ2agene prepared previously. Both the H and L chain transcription unitsincluded a cytomegalovirus (CMV) promoter (in place of themetallothionein promoter in the original reference) at the 5′ end and, apolyadenylation site at the 3′ end.

A similar vector (pNC-p40) was constructed for expression of the freep40 subunit which included a selectable marker gene (neomycin resistantgene) but still used the CMV promoter for transcription. The codingregion in this case included the natural leader sequence of the p40subunit for proper trafficking to the endoplasmic reticulum and assemblywith the fusion protein. Plasmid pNC-p40 was electroporated into cells,and cells were plated and selected in G418-containing medium. In thiscase, culture supernatants from drug-resistant clones were tested byELISA for production of p40 subunit.

The pdHL7-huKS-muγ2a-p35 expression vector was electroporated into theNS/0 cell line already expressing murine p40, as described in Gillies etal. (1998) J. Immunol. 160: 6195-6203. Transfected clones resistant tomethotrexate were tested for secretion of antibody determinants andmouse IL-12 by standard ELISA methods. The resulting protein waspurified by binding to, and elution from a protein A Sepharose column inaccordance with the manufacturers instructions.

Example 3 In Vitro Cytotoxic Activity of Combination Therapy

The cell lines engineered for use in animal models (example 1) weretested for their sensitivity to taxane-induced cytotoxicity in cellculture in the presence or absence of the an IL-2 based immunocytokineconsisting of the humanized form of the KS-1/4 antibody fused at thecarboxyl terminus of the H chain to human IL-2 (huKS-huγ1-huIL2,hereafter abbreviated, KS-IL2). Cells were seeded at 1000 cell/well in96 well flat-bottom plates and incubated for 24 hours at 37° C., 7% CO₂.Paclitaxel, at 2-fold dilutions from 200 ng/ml to 3.125 ng/ml, KS-IL2,at 200 ng/ml and IL-2, at 33.3 ng/ml (the equivalent amount of IL-2 inKS-IL2) were added in duplicate to the cell culture plates and incubatedfor 6 days at 37° C., 7% CO₂. The MTS colorimetric assay (Promega), ameasure of cell viability based on the cellular conversion of atetrazolium salt, was performed directly in the 96 well plates. Afterplates were read and recorded, viable adherent cells were stained withCrystal violet (Sigma, St. Louis, Mo.). Crystal violet stained plateswere used to verify MTS assay results. Results are expressed in tabularform. The IC₅₀ is the concentration of drug that produced cytotoxicityat a level of 50% of control.

A cytotoxicity assay was performed with paclitaxel (3 to 200 ng/ml)alone or combined with KS-IL2 (200 ng/ml) or IL-2 (33.3 ng/ml, theequivalent amount of IL-2 in KS-IL2) against CT26/KSA, LLC/KSA and4T1/KSA cells. There was little to no cytotoxicity of KS-IL2 or IL-2alone on the three cell lines tested (81% to 101% of control, Table 1).The addition of either KS-IL2 or IL-2 did not affect the cytotoxicity ofPaclitaxel. Therefore, since neither KS-IL2 nor IL-2 affects thecytotoxicity of paclitaxel, any enhancement in anti-tumor activity inmice by the combined treatments must be due to other mechanisms, whichoccur only in the tumor-bearing animal.

TABLE 1 Cytotoxicity of Paclitaxel in combination with IL-2 or KS-IL2CT26/KSA^(a) LLC/KSA^(b) 4T1/KSA^(b) Paclitaxel IC₅₀ (ng/ml) Taxol 27 616 Taxol + IL-2 (33 ng/ml) 30 8 20 Taxol + KS-IL2 (200 ng/ml) 26 5 19 %of Control IL-2 (33 ng/ml) 97 100 95 KS-IL2 (200 ng/ml) 90 101 81^(a)Average of three experiments ^(b)Average of two experiments

Example 4 Combination Therapy of LLC Skin Tumors with KS-IL2 and aTaxane

A tumor growth regression assay was performed using the aggressivelygrowing tumor, LLC/KSA, in which a single dose of paclitaxel (80 mg/kg)was followed one week later by KS-IL2 (20 μg) administered byintravenous tail vein injection for 5 days (FIG. 2). No effect of eitherthe paclitaxel or KS-IL2 given alone (on Days 0-4) was observed.However, when KS-IL2 was administered one week following paclitaxel, alarge reduction in average tumor volume (41% of control) and a tumorgrowth delay (TGD) of about 8 days was observed which was significantlydifferent than paclitaxel alone (p=0.023). No drug-related grosstoxicity was observed except for a<5% weight loss in the paclitaxeltreated groups.

Next, the effect of multiple doses of paclitaxel, generally considered amore effective chemotherapy schedule, was compared to a single dose ofpaclitaxel in combination with KS-IL2 to determine how the scheduleaffects the enhancement. KS-IL2 (20 μg, Days 0-4) alone again had noeffect on LLC/KSA tumor growth but paclitaxel alone, when given inmultiple doses (50 mg/kg, every other day), reduced the average tumorvolume to 63% of control and caused a 4 day tumor growth delay (TGD)(FIG. 3). When the KS-IL2 immunocytokine was administered one weekfollowing paclitaxel treatment, a reduction in tumor volume to 27% ofcontrol and a TGD of 10 days was observed which was significantlydifferent than paclitaxel alone (p=0.016). No drug-related grosstoxicity was observed except for a<5% weight loss in the paclitaxeltreated groups. The combined therapy group had even less weight loss.These positive combination therapy results are surprising consideringthe relatively short interval between chemotherapeutic (and potentiallyimmune damaging) treatment and the initiation of a treatment that isbased on the ability to stimulate lymphocyte proliferation andcytotoxicity.

One explanation for the combined effect is that taxane-induced apoptosisof a portion of the growing tumor mass reduced the interstitial pressurethat, in turn, increased the effective uptake of KS-IL2 into the tumor.Recent studies (Griffon-Etienne et al. 1999, CANCER RES. 59:3776-3782)indicate that the effect of a single dose of paclitaxel effectivelylowered interstitial fluid pressures with a maximum effect seen from 24to 48 hours (Griffon-Etienne et al. 1999, CANCER RES. 59:3776-3782).Although this may be the best time for uptake of the immunocytokine intothe tumor, it is also a very short time interval after chemotherapy.Nonetheless, we treated mice bearing LLC/KSA tumors with KS-IL2 for 5consecutive days beginning just 24 hr after receiving a single dose ofpaclitaxel. Results indicate that there is an even better combinedresponse when immunocytokine treatment was initiated earlier than a weekfollowing a single dose of paclitaxel with this tumor line as well ascolon carcinoma CT26 (see below).

Example 5 Combination Therapy of 4T1 Metastases with KS-IL2 and a Taxane

Since we found that treatment intervals between administration of ataxane and an immunocytokine could be shorter than expected, we testedcombination regimens in which the taxane and the immunocytokine aregiven on the same day and compared a single dose (75 mg/kg) ofpaclitaxel with a fractionated dose (25 mg/kg×3 days) given concurrentlywith KS-IL2 treatment (15 μg/dose×3 days given 4 hr after paclitaxel).For this experiment we used an experimental lung metastasis modelinduced with 4T1/KSA breast carcinoma cells. The doses of the drugs wereselected to be sub-optimal by themselves so that any potential additiveor synergistic activity could be observed.

Each agent given alone significantly (p<0.02) reduced average lungweights to a similar extent: 43% reduction for the single dose ofpaclitaxel, a 49% reduction for multiple doses of paclitaxel alone and a39% reduction with KS-IL2 alone (FIG. 4). The combination of paclitaxeland KS-IL2 further reduced lung metastases slightly but was less thanadditive: 58% reduction for single dose paclitaxel in combination withKS-IL2 and a 68% reduction for multiple dose paclitaxel in combinationwith KS-IL2. Even though no synergism was observed, the single dose ofpaclitaxel in combination with KS-IL2 resulted in a significantdifference compared to paclitaxel given alone (p=0.047).

Less than 10% weight loss was observed in all groups, however, thegreatest weight loss was obtained with 25 mg/kg of paclitaxel given 3times every other day. Based on these data, the best regimen in this 4T1lung metastasis assay with respect to the greatest effect of combinationtherapy was a single dose of paclitaxel followed by KS-IL2, as was thecase for the LLC/KSA tumor growth regression model. Since the dosinginterval in this case was only 4 hr, the results might not have beenoptimal for efficient tumor uptake.

Example 6 Combination Therapy of CT26 Skin Tumors with KS-IL2 and aTaxane

The results described in example 5 suggested that the time interval of 4hr between dosing the two agents might be too short. Perhaps the levelsof paclitaxel still remaining in the animal at the time of KS-IL2 dosingcould interfere directly with lymphocyte activation, thus reducing itspotential anti-tumor activity in the combination setting. Also, at the 4hr time point, the maximum effect on the tumor interstitial pressurewould not have been reached. Therefore, we designed another experiment,this time using established skin tumors of the CT26/KSA colon carcinoma,in which we combined a single dose of paclitaxel (75 mg/kg) with a 5-daycourse of KS-IL2 beginning 24 hr after administration of the taxane.Paclitaxel alone had no effect on tumor growth (FIG. 5). Treatment withsub-optimal doses of KS-IL2 (10 μg, Days 1-5) resulted in tumor volumesthat were 71% of control. A dramatic and synergistic reduction of tumorvolume to 8% of control was observed with the combination of paclitaxeland KS-IL2, which was significantly different from paclitaxel treatmentalone (p<0.001). A minimal weight loss of ˜5% was observed for bothpaclitaxel treated groups.

A second experiment was performed using the CT26/KSA model, this timetesting the effect of combined therapy on established liver metastasesand again using the 24 hr delay between paclitaxel administration andKS-IL2 treatment. We also compared the dose response of paclitaxel inthe combination therapy. Mice were injected with 25, 50, or 75 mg/kg ofpaclitaxel on Day 5 after metastasis induction, alone or followed oneday later with KS-IL2 (7 ug) for 5 days. A dose response effect wasobserved for paclitaxel alone, in which 25, 50, 75 mg/kg resulted intumor burdens of 49%, 23%, 10% of control, respectively (FIG. 6).Combining paclitaxel with KS-IL2 further reduced lung metastases to 12%,9%, and 6% of control for the same respective doses of paclitaxel. Thelowest dose of paclitaxel (25 mg/kg) in combination with KS-IL2 resultedin the greatest and most significant (p<0.001) reduction in tumor burdencompared to the higher doses of paclitaxel with KS-IL2. Therefore, thecombination of KS-IL2 preceded by paclitaxel resulted in a greateranti-tumor effect than either agent alone. Further, the lowest dose ofpaclitaxel in combination with KS-IL2 resulted in similar anti-tumorefficacy as the highest dose of paclitaxel alone. Hence, using a lowerdose of paclitaxel in combination with KS-IL2 would reduce toxicitywhile maintaining good efficacy.

Example 7 Measuring Uptake of KS-IL2 into Tumors

If the effect of single doses of cytotoxic drug treatment, prior toimmunocytokine therapy, is to decrease tumor interstitial pressure andincrease penetration of tumors, this should be measurable usingradioactively labeled immunocytokine, e.g. KS-IL2. Purified KS-IL2 waslabeled with ¹²⁵I by standard procedures (reference) through contract toa commercial vendor (New England Nuclear, Billerica, Mass.). Skin tumorsof CT26/KSA were implanted subcutaneously as described in Example 1 andallowed to grow until they reached from 100-200 mm³. Two groups of 4mice were injected with either paclitaxel (50 mg/kg) in vehicle orvehicle alone followed in 1 hr (Experiment 1) or 24 hr (Experiment 2) by10 μg of ¹²⁵I-KS-IL2 (95 μCi). Six hours after injecting theradiolabeled immunocytokine, the mice were sacrificed and their tumorswere surgically removed. As a control, livers of the animals were alsocollected and all tissues were weighed and then counted in a gammacounter. Results were expressed as the counts per minute (CPM) per gramof tissue by dividing the total CPM in the tissue by the weight.

When labeled KS-IL2 was injected 1 hr after paclitaxel treatment (FIG.7A), only a small increase in the amount of radioactivity was seen inthe excised tumors from animals receiving the drug. In contrast, whenlabeled KS-IL2 was injected 24 hr after paclitaxel treatment, a dramaticincrease in uptake was seen (>200 percent) relative to the vehiclecontrol (FIG. 7B). This great difference in tumor uptake between the 1hr and 24 hr time points is in agreement with the data on taxane-inducedchanges in interstitial pressure (Griffon-Etienne et al. 1999, CANCERRES. 59:3776-3782), and is consistent with the data in our tumor modelsshowing that treatment beginning 24 hr after paclitaxel is moreefficient than treatment at earlier times (4 hr).

We also tested whether other classes of drugs could increase the uptakeof labeled immunocytokine into solid tumors. In this case, mice wereinjected with a single dose of cyclophosphamide (40 mg/kg) either 24 hror 3 days prior to the experiment. ¹²⁵I-labeled KS-IL2 was injected intoall mice, including control mice pre-treated with PBS, and the amount ofradioactivity in excised tumors was determined 16 hr later. Results(FIG. 8) show that pre-treatment with cyclophosphamide increased theuptake of KS-IL2 by 48% in mice pre-treated 24 hr earlier and by 70% inmice pre-treated for 3 days.

Example 8 Combination Therapy with huKS-huγ4-IL2 and a Taxane

New forms of immunocytokines have been described recently that haveincreased circulating half-lives and improved efficacy due to a reducedaffinity for Fc receptors (see Gillies et. 1999, CANCER RES.59:2159-2166). One representative of these improved IL-2immunocytokines, huKS-huγ4-IL2, was tested in combination therapy with asingle dose of paclitaxel. Again, there was improved efficacy when thetwo drugs were given sequentially in mice bearing CT26/KSA skin tumors.

Example 9 Combination Therapy with huKS-muγ2a-muIL12 and a Taxane

In order to test whether the synergistic therapeutic effect is specificonly for IL-2 based immunocytokines, we treated established CT26/KSAbulky tumors first with paclitaxel (single dose of 75 mg/kg) followed 24hr later with a 5-day course of huKS-muγ2a-muIL12 (5 μg per day). Thisimmunocytokine represents a fusion between the murine form of the HuKSantibody (i.e. the constant regions were reverted to murine C kappa andC gamma 2a) and murine IL-12. It was necessary to use murine IL-12sequences because, unlike IL-2, this cytokine is highly species specificand the human form is not very active in the mouse. Results show thattreatment with paclitaxel alone had very little effect on tumor growth.Treatment with sub-optimal doses of huKS-muγ2a-muIL12 had an anti-tumoreffect and this was increased in mice that were treated first with asingle dose of paclitaxel.

Example 10 Combination Therapy with huKS-IL2 and an Alkylating Agent

i. The improved therapeutic effect of the combination of huKS-IL2 withcyclophosphamide, a chemotherapy drug in the alkylating agent class, wasalso demonstrated. 4T1 breast carcinoma cells were injectedintravenously into immuno-competent mice to establish pulmonarymetastases 3 days before treatment. Mice were treated with a single doseof cyclophosphamide (15, 40, or 80 mg/kg) followed three days later witha 5-day course of huKS-IL2 (15 ug/day). Even though the two lowest dosesalone caused only a modest reduction in lung metastasis tumor burden,the combination with huKS-IL2 resulted in a significantly large decreasein tumor burden compared to cyclophosphamide alone (p<0.05, FIG. 9).However, at the highest dose (80 mg/kg) no synergy occurs.

ii. The improved therapeutic effect of the combination of huKS-IL2 withcyclophosphamide was also demonstrated in a tumor growth assay, inimmuno-competent mice bearing established breast carcinoma subcutaneoustumors. Mice were treated with a single dose of 80 mg/kgcyclophosphamide, either alone or in combination with 5 daily doses ofhuKS-IL2 (30 μg) 3 days following the cyclophosphamide treatment.Average tumor volumes for huKS-IL2 and 80 mg/kg of cyclophosphamidealone were reduced by 31% and 69%, respectively (FIG. 9B). Thecombination treatment reduced average tumor volumes by 100% on Day 25which was significantly different than either huKS-IL2 alone orcyclophosphamide alone (p<0.05) and completely eliminated tumors in sixout of eight mice up to at twelve weeks after the initial treatment.Animals tolerated these treatments well with less than 10% weight lossobserved in all groups.

iii. The improved therapeutic effect of the combination of huKS-IL2 withcyclophosphamide was also demonstrated in a tumor growth assay, inimmuno-competent mice bearing established lung carcinoma subcutaneoustumors. Mice were treated with a single dose of 80 mg/kgcyclophosphamide, either alone or in combination with 5 daily doses ofhuKS-IL2 (20 μg) 3 days following the cyclophosphamide treatment.Average tumor volumes for huKS-IL2 and 80 mg/kg of cyclophosphamidealone were reduced by 2% and 27%, respectively (FIG. 9C). Thecombination treatment reduced average tumor volumes by 48% on Day 20which was significantly different than either huKS-IL2 alone orcyclophosphamide alone (p<0.05). Animals tolerated these treatments wellwith less than 10% weight loss observed in all groups.

Example 11 Combination Therapy with huKS-IL2 and an Alkylating Agent

The improved therapeutic effect of the combination of huKS-IL2 withCarboplatin, another chemotherapy agent in the alkylating agent class,was demonstrated. Mice bearing established non-small cell lung carcinomasubcutaneous tumors (LLC/KSA) were treated with Carboplatin (75 mg/kg)on Day 0 followed by three days later with a 5-day course of KS-IL2 (20ug per day). Carboplatin and KS-IL2 treatment alone each resulted in amodest decrease in tumor growth, however, only the combination treatmentsignificantly reduced the average tumor volume on Day 20 (p<0.05, FIG.10). Further, the growth of tumors in which mice were treated with thecombination compared to Carboplatin treatment alone was significantlydifferent (p<0.05).

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather then limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes which come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

Each of the patent documents and scientific publications disclosedhereinabove is incorporated by reference herein.

1. A method of inducing a cytocidal immune response against a tumor in amammal, the method comprising the steps of administering to a mammal:(i) an immunocytokine comprising an antibody binding site and acytokine; and, (ii) an immunocytokine uptake enhancing agent thatenhances an immune response induced by the immunocytokine.
 2. The methodof claim 1, wherein the antibody binding site binds to a cancer cell. 3.The method of claim 1, wherein the antibody binding site binds to atumor specific antigen.
 4. The method of claim 1, wherein theimmunocytokine uptake enhancing agent is co-administered with theimmunocytokine.
 5. The method of claim 1, wherein the immunocytokineuptake enhancing agent is administered prior to administration of theimmunocytokine.
 6. The method of claim 1, wherein the antibody bindingsite comprises, in an amino-terminal to carboxy-terminal direction, animmunoglobulin variable region, a CH1 domain, and a CH2 domain.
 7. Themethod of claim 6, wherein the antibody binding site further comprises aCH3 domain attached to the carboxy terminal end of the CH2 domain. 8.The method of claim 1, wherein the immunocytokine is a fusion proteincomprising, in an amino-terminal to carboxy-terminal direction, (i) theantibody binding site comprising an immunoglobulin variable regioncapable of binding a cell surface antigen on a preselected cell type, animmunoglobulin CH1 domain, an immunoglobulin CH2 domain, and (ii) thecytokine.
 9. The method of claim 8, wherein the antibody binding sitefurther comprises a CH3 domain interposed between the CH2 domain and thecytokine.
 10. The method of claim 1, wherein the cytokine of theimmunocytokine is selected from the group consisting of a tumor necrosisfactor, an interleukin, a colony stimulating factor, and a lymphokine.11. The method of claim 1, wherein said immunocytokine uptake enhancingagent is a taxane.
 12. The method of claim 11, wherein said taxane isselected from the group consisting of Taxol, docetaxel, 10-deacetylBaccatin III, and derivatives thereof.
 13. The method of claim 1,wherein said immunocytokine uptake enhancing agent is an alkylatingchemotherapeutic agent.
 14. The method of claim 13, wherein saidalkylating chemotherapeutic agent is selected from the group consistingof cyclophosphamide, carboplatin, and derivatives thereof.
 15. Themethod of claim 1, wherein two or more different immunocytokine uptakeenhancing agents are administered to said mammal.
 16. The method ofclaim 1, wherein two or more different immunocytokines are administeredto said mammal.
 17. The method of claim 1, wherein said immunocytokineuptake enhancing agent is administered about 24 hours before saidimmunocytokine.
 18. A composition for inducing an immune responseagainst a tumor in a mammal, the composition comprising: (i) animmunocytokine comprising an antibody binding site and a cytokine; and,(ii) an immunocytokine uptake enhancing agent.
 19. The composition ofclaim 18, wherein the antibody binding site comprises in anamino-terminal to carboxy-terminal direction, an immunoglobulin variableregion, a CH1 domain and a CH2 domain.
 20. The composition of claim 19,wherein the antibody binding site further comprises a CH3 domainattached to the C-terminal end of the CH2 domain. 21-27. (canceled)